Normal serum contains a number of lipoprotein particles which are characterized according to their density, namely, chylomicrons, VLDL, LDL and HDL. They are composed of free and esterified cholesterol, triglycerides, phospholipids, several other minor lipid components, and protein. Low density lipoprotein (LDL) transports lipid soluble materials to the cells in the body, while high density lipoprotein (HDL) transports these materials to the liver for elimination. Normally, these lipoproteins are in balance, ensuring proper delivery and removal of lipid soluble materials. Abnormally low HDL can cause a number of diseased states as well as constitute a secondary complication in others.
Under normal conditions, a natural HDL particle is a solid with its surface covered by a phospholipid bilayer that encloses a hydrophobic core. Apolipoprotein A-I and A-II attach to the surface by interaction of the hydrophobic face of their alpha helical domains. In its nascent or newly secreted form the particle is disk-shaped and accepts free cholesterol into its bilayer. Cholesterol is esterified by the action of lecithin:cholesterol acyltransferase (LCAT) and is moved into the center of the disk. The movement of cholesterol ester to the center is the result of space limitations within the bilayer. The HDL particle "inflates" to a spheroidal particle as more and more cholesterol is esterified and moved to the center. Cholesterol ester and other water insoluble lipids which collect in the "inflated core" of the HDL are then cleared by the liver.
Jonas et al., Meth. Enzym. 128A: 553-582 (1986) have produced a wide variety of reconstituted particles resembling HDL. The technique involves the isolation and dilapidation of HDL by standard methods (Hatch et al., Adv. Lip. Res. 6: 1-68 (1968); Scanu et al., Anal. Biochem. 44: 576-588 (1971) to obtain apo-HDL proteins. The apoproteins are fractionated and reconstituted with phospholipid and with or without cholesterol using detergent dialysis.
Matz et al., J. Biol. Chem. 257(8): 4535-4540 (1982) describe a micelle of phosphatidylcholine, with apoliprotein A1. Various ratios of the two components are described, and it is suggested that the described method can be used to make other micelles. It is suggested as well to use the micelles as an enzyme substrate, or as a model for the HDL molecule. This paper does not, however discuss application of the micelles to cholesterol removal, nor does it give any suggestions as to diagnostic or therapeutic use.
Williams et al., Biochem. & Biophys. Acta 875: 183-194 (1986) teach phospholipid liposomes introduced to plasma which pick up apoproteins and cholesterol. Liposomes are disclosed, which pick up apoprotein in vivo, as well as cholesterol, and it is suggested that the uptake of cholesterol is enhanced in phospholipid liposomes which have interacted with, and picked up apoproteins.
Williams et al., Persp. Biol. & Med. 27(3): 417-431 1984) discuss lecithin liposomes as removing cholesterol. The paper summarizes earlier work showing that liposomes which contain apoproteins remove cholesterol form cells in vitro more effectively than liposomes which do not contain it. They do not discuss in vivo use of apoprotein containing liposomes or micelles, and counsel caution in any in vivo work with liposomes.
It is important to note that there is a clear and significant difference between the particles of the present invention, and the liposomes and micelles described in the prior art. The latter involve a bilayer structure of lipid-containing molecules, surrounding an internal space. The construction of liposomes and micelles precludes filling the internal space, however, and any molecular uptake is limited to the space defined between the two lipid layers. As a result, there is much less volume available for pick up and discharge of materials such as cholesterol and other lipid soluble materials than there is for the particles of this invention, which expand in a fashion similar to a balloon, with interior space filling with the material of choice.
Anantharamaiah, in Segrest et al., Meth. Enzymol. 128: 627-647 (1986) describe a series of peptides which form "helical wheels", as a result of the interaction of the amino acids in the peptide with each other. Such helical wheels present a nonpolar face, and a polar face in their configuration.
Endotoxic shock is a condition, often fatal, provoked by the outer membrane of most gram negative bacteria (e.g., Escherichia coli; Salmonella typhimurium). The structure of the bacterial outer membrane has been fairly well elucidated, and a unique molecule, referred to as lipid A, which is linked to acyl chains via lipid A molecule's glucosamine backbone. See Raetz, Ann. Rev. Biochem. 59: 129-170 (1990) in this regard.
The lipid A molecule serves as membrane anchor of a lipopolysaccharide structure ("LPS") and it is the LPS which is implicated in the development of endotoxic shock. It should be pointed out that LPS molecules are characterized by a lipid A type structure and a polysaccharide portion. This latter moiety may vary in molecular details in different LPS molecules, but it will retain the general structural motifs characteristic of endotoxins. It would be incorrect to say that the LPS molecule is the same from bacteria to bacteria (see Raetz, supra). It is common in the art to refer to the various LPS molecules as "endotoxins", and this term will be used hereafter to refer to LPS molecules collectively.
In U.S. Pat. No. 5,128,318 the disclosure of which is incorporated by reference, it was taught that reconstituted particles containing both an HDL associated apolipoprotein and a lipid capable of binding an endotoxin to inactivate it could be used as effective materials for alleviating endotoxin caused toxicity.
It has now been found that various other materials may be used to treat endotoxin caused toxicity. Specifically, it has been found that apolipoproteins are not required in reconstituted particles, and that the reconstituted particle may contain a peptide and a lipid as defined supra, wherein the peptide is not an apolipoprotein.
It has also been found that endotoxin caused toxicity may be treated via sequential administration of either an apolipoprotein or a peptide followed by a lipid as described supra. It appears that following sequential administration the components assemble as a reconstituted particle and then act to remove endotoxin.
It has also been found that at least some individuals possess native levels of apoliprotein which are higher than normal levels such that effective endotoxemia therapy may be effectuated by administering reconstituted particles containing no apolipoprotein or peptide, but containing the lipid described supra.
In addition, the invention involves the use of the reconstituted particles and the components discussed herein for prophylaxis against endotoxin caused toxicity, by administering prophylactically effective amounts to subjects in need of prophylaxis.
These and other aspects of the invention are described in the disclosure which follows.